Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at –15°C) was induced when 50 M ABA or 3.2 M mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at –15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.Abbreviations ABA abscisic acid - MFD mefluidide - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ killing temperature for 50% of the embryos     

Somaclonal variation for freezing tolerance in a population derived from norstar winter wheat

Summary Progeny of 66 plants regenerated from callus cultures derived from immature embryos of Norstar winter wheat were evaluated as seedlings for tolerance to controlled freezing. Greater freezing tolerance than the parent cultivar was observed in both R₂ and R₃ regenerate families. LT₅₀ values (predicted temperatures at which mean survival frequencies are 50%) for four families in the R₂ generation and three families in the R₃ were significantly lower than that of Norstar. In both R₂ and R₃ generations, most families did not differ significantly from the cultivar Norstar, by three separate measures of tolerance. Significant variation among families was observed in both R₂ and R₃ generations for survival, but not for plant height. Variation within family in the R₃ generation was also significant, though smaller than that among families. In the R₃ generation, eighteen families were significantly less freezing tolerant than Norstar according to LT₅₀, while thirteen were significantly less tolerant according to survival at a minimum temperature of-17 °C.NRCC No. 28387. This publication describes research performed as part of the program of the Plant Biotechnology Institute of the National Research Council of Canada through Plant Biotechnology Fund contract No. 31964-4-0021 awarded to T. H. H. Chen and M. D. Lazar, Alberta Research Council, Edmonton, Canada     

Membrane alterations in winter rye and Brassica napus cells during lethal freezing and plasmolysis

Abstract Cells fixed during freezing or plasmolysis were used to study membrane alterations in hardened and non-hardened Brassica napus suspension-cultured cells and rye leaf mesophyll cells. The plasmalemma in non-hardened rye mesophyll cells formed multilamellar vesicles during lethal freezing at high subzero temperatures (–5°C). These vesicles became highly condensed at lower subzero temperatures (–10°C). Conversely, cold-hardened rye mesophyll cells did not undergo membrane alterations at these temperatures. The results from plasmolysis of B. napus and rye mesophyll cells hardened by ABA at 25 °C and low temperature (2°C), respectively, verify the cell response to lethal freezing. Again there was a continuum of responses with 1 kmol m^?3 balanced salt causing multilamellar protrusions. Appression of the plasmalemma against the tonoplast to form multilamellar vesicles and the invagination of these vesicles into the tonoplast were also observed in rye cells undergoing lethal plasmolysis. Increasing the plasmolysing solution to 3 kmol m^?3 occasionally caused the formation of multilamellar vesicles on the cell surface of hardened rye mesophyll cells.     

Characterization of a partial male fertility derived from Ogura-cms in progeny of a resynthesized Brassica napus

Summary The inheritance of a partial male fertile phenotype in somatic hybrid B. napus plants that carried novel mtDNA was investigated over five backcross generations to B. napus Triton. The recurrent parent and the original somatic hybrid both contained chloroplasts resistant to atrazine. The F₁ population contained mainly plants that were partial fertile, and some of the plants differed in mtDNA. The partial fertility predominated in the progeny of each backcross generation, but fully male sterile and fertile plants were also obtained. However, the sterility/fertility of these latter plants was not stable; both the fully male sterile and the male fertile plants produced progeny that were again predominantly partial male fertile. This pattern of predominant partial fertility but occasional sterile and fertile plants persisted in different nuclear backgrounds. Neither the male sterility nor the male fertility could be fixed and made stable. Test crosses indicated that restorer genes were probably not associated with appearance of male fertile plants. The evidence indicates that the behavior of the partial male fertility is cytoplasmic, and probably controlled by the chondriome.     

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.     

Mapping loci controlling vernalization requirement and flowering time in Brassica napus

Rapeseed cultivars (Brassica napus L.) can be classified into annual and biennial groups according to their requirement for vernalization in order to induce flowering. The genetic control of these phenotypic differences is not well understood, but this information could be valuable for the design of breeding approaches to accelerate rapeseed improvement. In order to map loci controlling this variation, a doubled haploid population, derived from a cross between annual and biennial cultivars, was evaluated for vernalization requirement and days-to-flowering in a replicated field experiment using three treatments: no vernalization, 4 weeks of vernalization and 8 weeks of vernalization. A linkage map of 132 RFLP loci was used to locate loci controlling these traits. Marker segregation in one region of linkage group 9 was strongly associated with the annual/biennial growth habit in the unvernalized treatment and with days-to-flowering in all three treatments. Two other regions with smaller effects on days-to-flowering were also identified.     

Characterization of a new myrosinase in Brassica napus

A full-length cDNA clone defining the new myrosinase gene family MC in Brassica napus was isolated and sequenced. Southern hybridization showed that the MC family probably consists of 3 or 4 genes in B. napus. MC genes are expressed in the developing seed, but not in the vegetative tissues investigated. In situ hybridizations to developing seeds showed that the MC genes are expressed in the myrosin cells of the embryo axis and the cotyledons. Complexes with myrosinase and myrosinase-binding protein (MBP) were purified and characterized. Sequencing of peptides from myrosinases occurring in the complexes showed that the 70 kDa myrosinase is encoded by the MC genes, whereas the 65 kDa myrosinase is encoded by the MB genes. This is in contrast to the 75 kDa myrosinase which occurs in free form and is encoded by the MA genes. Deglycosylations of the myrosinase complexes and the free myrosinase showed that the molecular sizes of the myrosinases could be reduced significantly by this treatment, and that the size differences between the different myrosinases are mainly due to differences in glycosylation.     

Differentiation of freezing tolerance and vernalization responses in Cruciferae exposed to a low temperature

High levels of freezing tolerance were induced in leaves of different Cruciferae species including Brassica napus, Arabidopsis thaliana, Barbarea vulgaris, Thlaspi arvenses and Descurainia sophia by low-temperature acclimation. Concomitantly, the amount of total RNA doubled in these three species. Analyses of methylation patterns and dosage of rRNA genes were carried out to determine whether or not alterations occur in this DNA during development of freezing tolerance. Hybridizations of Southern transfers with an rDNA probe revealed two additional EcoRI sites in purified DNA isolated from freezing-tolerant leaves of winter B. napus cv Jet Neuf and D. sophia (both of which require low temperatures for vernalization), but not in isolates of A. thaliana or spring B. napus cv Topas. An increase of rDNA cistrons was also observed in both B. napus cv Jet Neuf and D. sophia but not in A. thaliana or B. napus cv Topas upon cold acclimation. These results suggest that low temperature induced amplification of rDNA and the differential methylation of EcoRI sites may possibly be related to the vernalization process but may not be related to the development of freezing tolerance. However, the higher activity of RNA polymerase (2.5 times more) observed upon cold acclimation may explain the concomitant increase in total RNA and may be related to the development of freezing tolerance in the Cruciferae.     

Characterization of simple sequence repeat markers derived in silico from Brassica rapa bacterial artificial chromosome sequences and their application in Brassica napus

A collaborative Brassica rapa genome sequencing project is currently in progress to aid the identification of agronomically important traits in Brassica species. As an initial stage, the ends of over 110 000 bacterial artificial chromosome clones were sequenced and mined for simple sequence repeats (SSRs). We present the characterization of 40 of these SSRs and their application in Brassica napus. The markers were screened against six Brassica species and Arabidopsis, and demonstrated reliable amplification, genome specificity, cross‐amplification and significant polymorphism. These SSRs will be useful for genetic analysis of Brassica germplasm.     

Towards artificial seeds from microspore derived embryos of Brassica napus

Plant Cell, Tissue and Organ Culture (PCTOC) - Microspore derived embryos are haploid and their immediate diploidization generates doubled haploid homozygous plants, whereas a normal breeding...     

Purification and Characterization of Methionine:Glyoxylate Aminotransferase from Brassica carinata and Brassica napus

The first step in the biosynthesis of allylglucosinolate from methionine in Brassica is thought to be the transamination of methionine to 2-keto-4-methylthiobutyrate. By using Q-Sepharose and Red Agarose, followed by high resolution anion exchange chromatography and chromatofocussing, a methionine:glyoxylate aminotransferase (MGAT) was purified to homogeneity from leaves of Brassica carinata var R-4218, and approximately 5000-fold from leaves of Brassica napus var Topas. The final purification was accomplished using nondenaturing polyacrylamide gel electrophoresis. The enzyme has a pl of 4.3, a native molecular mass of 230 to 290 kilodaltons, and a subunit molecular mass of approximately 50 kilodaltons. Four isozymes of the enzyme were identified in the six species of Brassica commonly cultivated. Nonglucosinolate producing species had only low levels of MGAT or an MGAT isozyme which was distinctly different from that in Brassica.     

Cytokinins in a genic male sterile line of Brassica napus

Endogenous cytokinin levels were analyzed in four different organs viz., root, stem, leaf and mature flower, of the wild type (cv. Westar) and a genic male sterile (GMS) line of Brassica napus . Various cytokinins viz., bases (Z, DZ), ribosides (ZR, DZR), glucosides (OGZ + OGZR, OGDZ + OGDZR) and nucleotides (ZNT, DZNT) were quantified using Amaranthus betacyanin bioassay and ELISA. The major cytokinin in all the organs was DZ. In general, leaves had the highest levels of cytokinins as compared to the other organs. Root, stem and mature flowers of the wild type plants had higher levels of cytokinins and their metabolites than the GMS line. However. levaves of the GMS line had greater amount of various cytokinins as compared to the wild type. The major cytokinin quantitatively different between the two lines was DZ. The results suggest the possible involvement of cytokinins in the expression of genic male sterility in Brassica napus .     

Characterization and Expression of a GDSL-Like Lipase Gene from Brassica napus in Nicotiana benthamiana

The GDSL esterase and lipase families play important roles in abiotic stress, pathogen defense, seed development and lipid metabolism. Identifying the lipase activity of the putative GDSL lipase is the prerequisite for dissecting its function. According to the sequence similarity and the conserved domains, we cloned the Brassica napus BnGLIP gene, which encodes a GDSL-like protein. We failed to identify the BnGLIP lipase activity in the bacterium and yeast expression systems. In this paper, we expressed the BnGLIP gene by fusing a 6× His tag in Nicotiana benthamiana and purified the recombinant protein. The extraction buffer contained 1 % (v/v) n-caprylic acid and was able to remove most of the protein impurities. About 50 μg of recombinant BnGLIP was obtained from 1 g of N. benthamiana leaves. The lipase activity was tested with the purified BnGLIP and the maximum enzyme activity reached 17.7 mM/mg. In conclusion, this study found that the recombinant protein BnGLIP expressed in tobacco system was effectively purified and was detected as a GDSL lipase.     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary Under an intermediate temperature regime (23° C/18° C; day/night), microsporogenesis in stamens of the ogu cytoplasmic male-sterile (CMS) line of Brassica napus terminated by the tetrad stage, although in some cases degeneration of the sporogenous tissue occurred prior to meiosis. In most cases the tetrads were collapsed and bounded by a sparse exine, but contained many organelles. Also, the tapetum in CMS anthers was abnormal and often highly vacuolated by the tetrad stage. Under low temperature conditions (18° C/15° C; day/night), neither microsporogenous nor tapetal tissues were observed. In the normal stamens, no differences were observed under different temperature regimes. In conjunction with the adjoining paper, this study demonstrates that temperature conditions strongly affect the cytological processes associated with microsporogenesis in the CMS anthers.     

The effects of phenotypic plasticity on photosynthetic performance in winter rye, winter wheat and Brassica napus

The contributions of phenotypic plasticity to photosynthetic performance in winter (cv Musketeer, cv Norstar) and spring (cv SR4A, cv Katepwa) rye (Secale cereale) and wheat (Triticum aestivum) cultivars grown at either 20°C [non‐acclimated (NA)] or 5°C [cold acclimated (CA)] were assessed. The 22–40% increase in light‐saturated rates of CO₂ assimilation in CA vs NA winter cereals were accounted for by phenotypic plasticity as indicated by the dwarf phenotype and increased specific leaf weight. However, phenotypic plasticity could not account for (1) the differential temperature sensitivity of CO₂ assimilation and photosynthetic electron transport, (2) the increased efficiency and light‐saturated rates of photosynthetic electron transport or (3) the decreased light sensitivity of excitation pressure and non‐photochemical quenching between NA and NA winter cultivars. Cold acclimation decreased photosynthetic performance of spring relative to winter cultivars. However, the differences in photosynthetic performances between CA winter and spring cultivars were dependent upon the basis on which photosynthetic performance was expressed. Overexpression of BNCBF17 in Brassica napus generally decreased the low temperature sensitivity (Q₁₀) of CO₂ assimilation and photosynthetic electron transport even though the latter had not been exposed to low temperature. Photosynthetic performance in wild type compared to the BNCBF17‐overexpressing transgenic B. napus indicated that CBFs/DREBs regulate not only freezing tolerance but also govern plant architecture, leaf anatomy and photosynthetic performance. The apparent positive and negative effects of cold acclimation on photosynthetic performance are discussed in terms of the apparent costs and benefits of phenotypic plasticity, winter survival and reproductive fitness.     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary In the ogu cytoplasmic male-sterile (CMS) line of Brassica napus, stamen morphology was influenced by temperature conditions. Under a high temperature regime (27° C/23° C; day/ night) CMS stamens had a near-normal morphology, but microsporogenesis proceeded to a maximum of the microspore stage. However, compared to the normal stamens, the occurrence of sporopollenin-like deposits in the tapetum and deposition of exine on the microspores was sparse. Also, the tapetal cells of the CMS line were often highly vacuolate and failed to degenerate at the same stage as the normal. Ultrastructural changes in the mitochondrial matrix and cristae plus dilation of the endoplasmic reticulum, which occurred during development in sporogenous tissues of the normal line, were often lacking or mistimed in the mutant. Due to extensive variation, even between adjacent locules, the cytological differences between the normal and CMS anthers cannot be ascribed as the cause of male sterility in the ogu CMS line of B. napus, rather they may be the consequence of it.